Field of Invention
The present invention relates to a microbial animal cell field, and more particularly to a human hepatoma cell line and applications thereof.
Description of Related Arts
Human liver has many important physiological functions including: synthesizing and secreting large amounts of serum proteins, such as albumin and lipoproteins; synthesizing and transporting lipids coupling to proteins; detoxification; synthesizing and secreting bile; synthesizing sugar to regulate human blood sugar; decomposing amino acid to synthesize urea; activating vitamin; synthesizing and decomposing glycogen; synthesizing glutathione and metallothionein, and other functions.
With the progress of biotechnology, the gene recombination and cell fusion technology can produce more useful biological products. The animal cell culture technology is even more important. Based on that the liver has more physiological functions than other organs, including the functions of synthesizing and secreting the large amounts of cytoplasmic proteins, the ability to synthesize albumin and lipoproteins, the ability to transport lipid, urea, glycogen, and glutathione, and the stronger metabolism ability, researchers have more interested to use the liver cell culture technology to produce the above useful biological products. Therefore, in order to explore the functions of the liver and produce related biological products, the liver cell culture technology is urgently needed. However, cultivation techniques are not satisfactory at present for maintaining hepatocytes (normal liver cells) capable of producing plasma proteins during cultivation periods. Normal human hepatocytes are extremely difficult to obtain due to their rapid decrease in viability following autopsy.
Additionally, the human liver is one of few organs in adults capable of regeneration. However, replicative cultures of adult human hepatocytes have never been adequately established, as these cells have a very limited lifespan when put into cell culture.
Some existing liver cell lines are derived from a non-human source or some sources having significant difference with human hepatocytes, so the existing liver cell lines cannot be widely used. Currently, some liver cell lines are derived from experimental animals, such as rat (Tsao et.al, Exp. Cell Res, 1984, 154:38-52; Enat et al., Proc. Natl. Acad. Sci., USA, 1984, 87:1411-1415), and some are delivered from liver epithelial cells in rat, which are cultured based on serum free medium (Chessebeuf and Padieu, InVitro, 1984, 20:780-795; Enat et al., Proc. Natl. Acad. Sci., 1984, 81:1411-1415). Rat liver cells have been transformed by transfection with SV40 DNA (Woodworth et al., Cancer Res., 1987, 46:4018-4026; Ledley et al., Proc. Nat. Acad. Sci. USA, 1987, 84:5335-5339). Those cells are not suitable for the study of human drug metabolism or hepatocarcinogenesis because of xenobiotic metabolism differences between rat and human liver cells.
In addition, related reports which introduces the asexual reproduction technology for the human liver cells have been introduced (Kaighn and Prince, Proc. Nat. Acad. Sci., 1971,68:2396-2400), and the long-term cultivation of the human fetal livers have also been established (Salas-Prato, M. et al., In Vitro Cell Dev. Biol., 1988, 24:230-238; Sells, M. A. et al., In Vitro Cell Dev. Biol., 1985,21:216-220). Since the metabolism between the fetal liver cells and adult liver cells are different, the fetal liver cells cannot be used for studying the formation of tumor and the application of toxicology. The adult liver cells are more suitable for studying the formation of tumor and the application of toxicology.
In order to find an alternative model as a study mode for liver cells, researchers have created cell lines derived from the liver cancer, which have the same function as normal hepatocytes (such as the function of the secretion of albumin). Currently, a variety of hepatoma cell lines have been established. (e.g., Knowles et al., U.S. Pat. No. 4,393,133, issued Jul. 12, 1983; Knowles B. B. et al., Science, 1980,209:497-499; Monjardino J. and Crawford E., Virology, 1979,96:652-655; Park J. G. et al., Int. J. Cancer, 1995, 62:276-282; Fuand Cheng, Antimicrobial Agents and Chemotherapy, 2000, 44 (12) :3402-3407), such as that the U.S patent of the hepatoma cell line HepG2(U.S. Pat. No. 4,393,133). Furthermore, studies related to the HepG2 have been reported. (Kelly et al., In Vitro Cell, and De. Biol, 1989, 25:217-222; U.S. Pat. No. 5, 290,684; and Darlington et al., In Vitro Cell, and Dev. Biol., 1987, 2-3:349-354). In addition, hepatoma cell line Huh7 has been established by Nakabayashi (Cancer Research, 1982, 42:3858-3863). According to the existing published literatures, the existing hepatoma cell lines include, but are not limited to: HLF (Okayama University, medical school :1975), HLE, c-1 (Okayama University, medical school: 1975), HuH-6clone5 (Okayama University, medical school: 1976), HuH-7 (Okayama University, medical school: 1979), C-HC-4 (Hokkaido University, school of medicine: 1979), HCC-M (Keio University, school of medicine: 1980), JHH-1 (The Tokyo Jikei University School of Medicine: 1980), JHH-2 (The Tokyo Jikei University School of Medicine: 1982), JHH-4 (The Tokyo Jikei University School of Medicine: 1983), KIM-1 (Kurume University, school of medicine: 1983), JHH-5 (The Tokyo Jikei University School of Medicine: 1984), JHH-6 (The Tokyo Jikei University School of Medicine: 1984), OHR (Showa University, school of medicine: 1985), KMCH-1 (Kurume University, school of medicine: 1985), KMG-A (Kurume University, school of medicine: 1985), JHH-7 (The Tokyo Jikei University School of Medicine: 1986), JHC-1 (The Tokyo Jikei University School of Medicine: 1986), KYN-1 (Kurume University, school of medicine: 1986), KYN-2 (Kurume University, school of medicine: 1987), HCC-T (Keio University, school of medicine: 1986), HPT-NT/D3 (Kyushu University, faculty of medicine: 1986), Hep-tabata (Mie University, Faculty of Medicine: 1986), HuCC-T1 (Toyama Medicine and Pharmaceutical University, faculty of medicine: 1987), HuH-28 (Okayama University, medical school: 1987). The relevant data can be found in HUMAN CELL, Vol. 1, No. 1, p. 106-126, 1988.
There have been a lot of research reports using the bioreactor to culture liver cells in order to manufacture artificial liver. The artificial technology has dramatically been improved in the progress of kidneys, heart and lung transplant. The use of artificial liver and other techniques to long-term remain the functions of liver have been reported (e.g., Anand A. C., Indian J. Gastroenterol., 2003,22 Supp12: S69_74; Ueda et al., ASAIOJ, 2003,49 (4) :401-6; Tilles et al., J. Hepatobiliary Pancreat. Surg., 2002,9 (6) :686-96; Metab. Brain Dis., 2005,20 (4) :327-35; and Park and Lee, J. Biosci Bioeng., 2005,99 (4) :311-9). Liver assist devices (LAD) have been described previously (e.g., Lu et al., Tissue Eng., 2005, 11 (11-12) :1667-77; Pless and Sauer, Transplant Proc., 2005,37 (9) :3893-5; Millis and Losanoff, Nat. Clin. Pract. Gastroenterol Hepatol., 2005,2 (9) :398-405; and George J., J. Assoc. Physicians India, 2004, 52:719-22).These liver assistance devices are often used in transplant surgery. For example, an artificial liver and liver assistance devices are able to make fulminant hepatic failure patients keep away and calm before surgery, and make the patients more stable after the transplant surgery, and more particularly when the patents have no response to perfusion, the artificial liver and the liver assistance devices may serve as a substitute for transplant in certain circumstances. In order to make better use of bio-artificial livers and liver assistance devices, the small and compact in size bioreactor are needed. Therefore, the cells cultured in the bioreactor are required to produce sufficient liver-specific proteins. Researchers have successfully cultured liver cells HepG2 in the above mentioned liver assistance devices cells to produce an anti-apoptotic protein Bcl-2 (Terada S., J. Bioscl. Bioeng., 2003, 95 (2) :146-51).
Liver cancer is a common cancer, wherein most of the liver cancers are caused by hepatitis viruses, such as hepatitis B virus (HBV) and hepatitis C virus (HCV). Currently, no effective therapy is able to cure this disease. HCV infection is a major human infectious disease, and there is no effective vaccine to prevent HCV infection. There have been parts of evidences introduced to prove an importance of the cellular immune in the clearance of hepatitis virus, but the antibody-mediated antiviral responses within the patients' bodies are not defined. Most of antibodies in the patient's body can neutralize viral antigens, but the levels and concentration of neutralized antibodies within the serum are unknown, such that the study of the neutralizing antibodies is limited due to the lack of the cell culture model and animal model supporting virus infection.
Despite some of the liver cell lines are derived from the human hepatoma cells, most of these existing cell lines are poorly differentiated and the functions thereof are not perfect. To our knowledge, there is no liver cell line which is able to support HBV and HCV infection, such that the development and application of antiviral drugs are limited. Although, some research teams use primary human hepatocytes and detect low-level replication of HCV therein, but there is no liver cell line supporting the strong replication of wild-type HCV. (Tagawa M. etal., J. Gastroenterol Hepatol, 1995, 10:523-527; Ito T. et al., J. Gen. Virol., 1996, 77 (Pt.5):1043-1054; Ito T et al., Hepatology, 2001, 34:566-572). In 2005, several groups reported the success of cell culture system that supports the lifecycle of one particular strain, JFH1, a genotype 2a isolate of HCV (Wakita T. et al., Nat. Med., 2005,11 (7) :791-796; Zhong J. et al., PNAs, 2005,102 (26) :9294-9; Lindenbach B. D. et al., Science2005, 309 (5734) :623-6).
Currently, the only successful animal model to support HCV infection is the chimpanzee (Lanford R. E. et al., Virology, 2002, 293:1-9). This animal model has the advantages of showing the lifecycle of the virus, although the pathology is different than the human with virus infection (Alter M. J. et al., N. Eng. J. Med., 1999, 341:556-562; Bassett S. E. et al., J Virol, 1998, 72:2589-2599), and this model provided tremendous knowledge about the host immune responses to HCV infection. The maximum limitation of the above mentioned model is that the source of chimpanzee is less and the cost is extremely expensive. Tupaia is a small animal which is close to the primates, so it is easy to adapt to the laboratory environment. Based on some studies, Tupaia is able to be infected by a variety of human viruses, including hepatitis virus (die Z. C.et al., Virology, 1998, 244:513-520).
Transgenic mice that express HCV core protein have been used to study the liver pathology and carcinogenesis (Lemon S. M. et al., Trans. Am. Clin. Climatol. Assoc., 2000, 111:146-156). Most of the transgenic mice cannot show the toxicity to hepatocytes, while one transgenic animal showed lymphocytic infiltration and hepatocyte necrosis (Zhao X. et al., J. Clin. Invest, 2002,109:221-232), and another two models suffer from the steatosis and hepatoma (Moriya K. et al., J. Gen. Virol, 1997,78 (Pt.7) :1527-1531; Moriya K. et al., Nat. Med., 1998,4:1065-1067). The HBV transgenic mice are able to be used for studying the mechanism of immune response to HBV (Chisari F. V. et al., Science, 1985, 230:1157-1160; Chisari F. V. et al., Hepatology, 1995, 22:1316-1325). However, transgenic models are not yet widely used in the studies on HCV immunology. One inherent problem using transgenic mouse models is host immune tolerance to viral proteins.
According to the above mentioned published research reports, liver cell lines and animal models for HBV and HCV are extremely needed. The potential applications of these cell lines include, but are not limited to: screening and evaluating anticancer drugs; culturing HBV and HCV in a manner resembling the naturally occurring infection; screening and evaluating antiviral drugs; studying metabolic functions of hepatocytes.